1. Technical Field
The application concerns a vaccine on the basis of allogeneic tumor cells for the therapeutic treatment of tumor diseases, as well as a method to make such vaccine. Furthermore, the application concerns transfected human tumor cells for use as a vaccine.
2. Background Information
Apart from conventional methods to treat cancerous diseases, such as radiation and chemotherapy, which since the 1950's comprise the only option to treat advanced and disseminated tumor diseases, immunotherapy seems to be a promising approach when confronted with metastasizing tumors.
Immunotherapy aims to enhance the natural response against the tumor disease by means of modifications derived from the repertoire of gene technology, thus increasing the “alertness” of the immune system against cancer cells and improving the defense reaction in such a way that the tumor is fought by the body itself.
Some malign diseases such as advanced renal cell carcinoma seem to be relatively sensitive to immunotherapeutic intervention, however systemic therapy with cytokines such as IL-2 and IFN-alpha is associated with sometimes harsh side effects. Hence, other immunotherapeutic protocols have been developed.
Most clinical studies currently rely on removal of the tumor, subsequent ex-vivo transfection of the tumor cells with a therapeutic gene, radiation of the tumor cell population and subsequent re-implantation of the modified tumor cells. By such tumor vaccination, the anti-tumor response can be increased in differing amounts, depending on the transfected therapeutic gene.
Based on this approach and results from studies in experimental animals, a number of clinical studies of phase I and II were approved (Finke et al., 1997; Wittig et al., 2001).
Preliminary results from these studies, however, indicated that apart from good tolerability of the therapy, partial or complete remissions were observed only in few cases.
Gene transfer of CD40L/CD154 was able to induce eradication of the tumor and immunity in a colon carcinoma mouse model (Sun et al., 2000).
The applicants/assignees of the present application were able to show that transfer of expression plasmids for human interleukin 7 (IL-7) into tumor cells leads to increased sensitivity against effector cells of the immune system, especially in autologous transfer (Finke et al., 1997, Cancer Gene Ther. 4: 260-268). Similarly, the inventors were able to demonstrate that after transfection of two therapeutic genes (IL-7, GM-CSF) into autologous tumor cells, a clinically significant response could be observed in 50% of the patients (WO 02/060476).
It is known from the teaching of U.S. Pat. No. 5,681,562 to inject cells that had been transfected with DNA or RNA encoding cytokines into patients suffering from certain cancers. The immune system of the patient is meant to be stimulated against tumor antigens. In the patent disclosure experiments are described in which murine fibroblasts are transfected by retroviral vectors coding for IL-2. An in-vivo experiment is also described, in which the efficacy of the treatment was tested in a murine colon carcinoma model. Mice that were injected with transfected fibroblasts subcutaneously, developed a significantly slower growth of tumor in comparison to control groups. In-vitro experiments with human transfected fibroblasts showed a significantly increased expression level of IL-2. Apart from the lack of clinical data beyond the mouse model, the viral vectors employed as expression vectors in these experiments are to be regarded as not advantageous. The instability of the attenuated vaccine strain does not allow exclusion of its reversion to a virulent strain, and the viral components can be immunogenic themselves, which results in a their efficacy being decreased by the immune system of the patient. These disadvantages, outlined here only as part of the disadvantages of the viral system, significantly compromise the use of the system as a vector in gene therapy.
Several publications show that the best therapeutic results are attained by combining cytokine genes with the growth factor GM-CSF (Paillard, 1998, Hum. Gene Ther. 9: 2457-2458; Schadendorf et al., 1995, J. Mol. Med. 73: 473-477). The importance of GM-CSF in tumor antigen vaccinations was also shown in the enhancement of the clinical and immunological anti tumor response to peptide vaccination (Jäger et al., 1996). It appears that the activation of antigen-presenting dendritic cells and the stimulation of an effector cell population has a pivotal role in this context.
So far, however, it is unclear what cytokine genes trigger the most effective anti-tumor response in combination with an immunogenic preparation and GM-CSF.
In experiments with mice, it was shown that vaccination with expression constructs encoding IL-7 results in an anti-tumor effect (Miller et al., 1993, Blood 18: 3686-3694; Murphy et al., 1993, J. Clin. Invest. 92: 1918-1924). It is also known that incubation of cytotoxic lymphocytes (CTL) with IL-7 or IL-7 transfected cells leads to tumor regression in mice (Jicha et al., 1991; Hock et al., 1993), and the transfection with IL-7 and B7.1/CD80 leads to infiltration of CD28+CD25+ T-cells and immunity (Cayeux et al., 1995).
So far, no therapeutic success has been attained, not even in a mouse model.
Furthermore, in experiments relating to the stimulation of immune responses by gene therapy with plasmid DNA or oligonucleotide sequences, it was observed that certain nucleic acid sequences that comprise CpG motifs (CpG=unmethylated cytosine-guanosine dinucleotide) can have an enormous immune stimulatory effect (Schmidt-Wolf et al., 1989, J. Immunol. Methods 125: 185-189). Immune stimulatory sequences (ISS) have been employed very early as adjuvants in DNA based immunization protocols against infectious pathogens for this reason (Sato et al., 1996, Science 273: 352-354). In experiments on mice, it was demonstrated that injection of CpG-rich DNA sequences leads to a strong activation of B-cells and stimulates the expression of certain cytokines, e.g. IL-6 and GM-CSF. It was also shown in a mouse model that immunization with CpG oligodeoxyribonucleosides (ODN) together with a fusion protein three days prior to tumor inoculation could prevent tumor growth in the mouse (Liu et al., 1998). The teaching of use and synthesis of immune stimulatory CpG-containing ISS is explained in detail in WO 98/18810.
From WO 00/04918 it is known to transfect tumor cells with genes encoding, for example, interferon gamma and GM-CSF. As expression vectors, plasmids are used. This application discloses a concept to treat tumor disease by immune therapy. Relevant in-vivo or in-vitro data or clinical results proving the efficacy of the claimed vaccine are not shown. It should be added that these plasmid-based vectors are not useful without restriction for their employment in human gene therapy, since they comprise genetically functional units that are necessary for their replication, apart from the therapeutic sequences. They also comprise antibiotic resistance genes needed for their selection. The result is a continuous expression of therapeutically not desirable mammalian or bacterial proteins.
Despite research over many years and promising approaches it has not been possible to develop an efficacious therapy on the basis of immune functions against tumor diseases by applying immunogenic substances.